Immobilization of a protein has been widely applied in diagnosis and other fields such as biosensor or enzyme reactor. As methods to immobilize a protein, chemical cross-linking using a cross-linking agent such as glutaraldehyde, physical adsorption using hydrophobic binding, and chemical coupling using amine reactive group have been known. The biggest problem that these methods have to overcome is to maintain the activity of a protein or polypeptide supposed to be immobilized. For the chemical cross-linking or coupling, the activity might be inhibited by new chemical bonds or changes in chemical properties of a target protein by such bonds. For the physical adsorption, when a target protein is adsorbed on the hydrophobic surface directly, the structure of the protein might be changed to cause denaturation. To solve the above problems, immobilization using a linker has been tried, in which a linker binds to a polypeptide, the target of immobilization, as an immobilization mediator (Lee J. M. et al., Anal. Chem. 79: 2680-2687, 2007; Ogiwara K. et al., Biochem. Biophy. Res. Comm. 345: 255-259, 2006; Nagaoka M. et al., PLoS ONE 1: e15, 2006). The most representative method to bind a linker to a biologically active polypeptide is to synthesize a recombinant fusion protein based on genetic engineering techniques. For example, glutathione molecule capable of recognizing glutathione-S-transferase (GST) is pre-conjugated on the surface for enzyme-linked immunosorbent assay (ELISA) (Sehr P. et al., J. Immuno. Methods. 253: 153-162, 2001). However, this method is not effective for non-reactive hydrophobic surface such as polystyrene because glutathione is difficult to bind to such non-reactive hydrophobic surface.
In the meantime, another method has been made using a linker having a hydrophobic domain, which was to increase efficiency in adsorption of a protein and at the same time to prevent denaturation thereof so as to maintain biological functions of the target protein after adsorption and thus to be ready for use as the surface for cell culture. A linker for the fusion protein immobilization based on hydrophobic binding is selected among those proteins having a strong hydrophobicity such as Fc domain of immunoglobulin (Ogiwara K. et al., Biotech. Letters 27: 1633-1637, 2005; Nagaoka M. et al., PLoS ONE 1: e15, 2006) and hydrophobin (Qin M. et al., Colloids Surfaces 60: 243-249, 2007). This linker facilitates adsorption without requiring a pre-surface treatment.
For example, EGF-Fc or cadherin-Fc is fixed on a polystyrene culture dish to prepare EGF or cadherin adsorbed surface, and then epithelial tumor cells or embryonic stem cells are cultured thereon. Then, the cells demonstrate different biochemical and cell-biological characteristics (Ogiwara K. et al., Biotech. Letters 27: 1633-1637, 2005; Nagaoka M. et al., PLoS ONE 1: e15, 2006). Alternative binding methods have been reported. For example, U.S. Patent No. 20040235050 describes a protein immobilization method using hydrophobin for the conjugation of an enzyme onto the hydrophobic surface. However, when Fc and hydrophobin are used, carboxy-terminal of Fc and hydrophobin is necessarily adsorbed onto the hydrophobic surface and then amino terminal of Fc and hydrophobin has to bind with a biologically active target polypeptide. So, if it is the case that carboxy terminal is necessary for the activation of a biologically active target polypeptide, these methods are not appropriated. In addition, the linker region of a fusion protein for hydrophobic binding is mostly originated from animals and fungi, so that when it is expressed in E. coli and purified from it, inclusion body formation would be a problem.
Thus, the present inventors tried to solve the problems of the conventional methods. As a result, the present inventors completed this invention by confirming that a biologically active polypeptide fusion protein can be prepared by using maltose binding protein facilitating the expression and purification in E. coli owing to its excellent maltose binding capacity, and when this fusion protein is fixed by simple physical adsorption on the hydrophobic surface such as polystyrene by using the hydrophobic domain of maltose binding protein (Fox J. D. et al., Protein Science 10: 622-630, 2001), the biologically active polypeptide of the fusion protein still retains its biological activities.